What is first strand synthesis?
Recommendations. Recommendations. The SuperScript™ First-Strand Synthesis System for RT-PCR is optimized to synthesize first-strand cDNA from purified poly(A)+ or total RNA. The system can be used with as little as 1 ng or as much as 5 µg of total RNA.
What is 5X first strand buffer?
First Strand Buffer, 5X, Teknova. Nucleic Acid Reagents RT-PCR and cDNA Reagents. First strand synthesis buffer designed for use in reverse transcription. Consists of Tris-HCl (250 mM), pH 8.3, potassium chloride (375 mM), magnesium chloride (15 mM) and DTT (50 mM).
How is the second strand of cDNA synthesized?
The second strand is synthesized using oligo(dA) as a primer. Several oligo(dA) molecules probably anneal to the poly(dT) tract. Because the 3′ exonuclease activity of T7 DNA polymerase is very high, the region of the tract annealed to these oligo(dA) molecules is digested.
What is first strand cDNA synthesis?
First-strand cDNA synthesis can be primed using random hexamers, oligo(dT), or gene-specific primers (GSPs): Random hexamers are the most nonspecific priming method, and are typically used for difficult or high GC-content mRNA.
Is the 1st strand cDNA synthesis reaction an amplification step?
The first-strand cDNA from the synthesis reaction may be amplified directly using PCR. We recommend using 10% of the first-strand reaction (2 μl) for PCR. However, for some targets, increasing the amount of first-strand reaction to up to 10 μl may result in increased product yield.
Why is an oligo dT primer used during synthesis of the first DNA strand of cDNA?
Oligo (dT)18 Primer is suitable for use as a primer for first strand cDNA synthesis with a reverse transcriptase. The primer hybridizes to the poly-adenylated tail found on the 3´ end of most eukaryotic mRNAs. Oligo (dT)18 ensures that the 3´ end of mRNAs are represented.
How is cDNA synthesized?
In molecular biology, complementary DNA (cDNA) is synthesised from an RNA template in a reaction catalysed by the enzyme reverse transcriptase (RTase). cDNA synthesis is the first step in many molecular biology workflows, such as gene expression studies using real-time PCR.
Is second strand synthesis necessary?
You don’t need a separate 2nd strand reaction. Your forward PCR primer will synthesize the 2nd strand during the 1st cycle of amplification.
What is the best way to synthesize first strand cDNA?
For first strand cDNA synthesis using total RNA or poly(A)+-selected RNA primed with oligo(dT), random primers, or a gene-specific primer. Important guidelines Pre-warm the 5× SSIV Buffer to room temperature before use. Vortex and briefly centrifuge the buffer prior to preparing the reverse transcription reaction mix. Online resources
What is the best primer for first strand cDNA synthesis?
For first strand cDNA synthesis using total RNA or poly(A)+-selected RNA primed with oligo(dT), random primers, or a gene-specific primer. Important guidelines Pre-warm the 5× SSIV Buffer to room temperature before use.
How is cDNA synthesized using the superscript® III first strand system?
Using the SuperScript® III First-Strand System. cDNA synthesis is performed in the first step using either total RNA or poly(A)+-selected RNA primed with oligo(dT), random primers, or a gene-specific primer. In the second step, PCR is performed in a separate tube using primers specific for the gene of interest.
What is the superscript III first-strand synthesis system for RT-PCR?
The SuperScript® III First-Strand Synthesis System for RT-PCR is optimized to synthesize first-strand cDNA from purified poly(A) + or total RNA. RNA targets from 100 bp to >12 kb can be detected with this system.